New chemical tools for probing activity and inhibition of the NAD+ -dependent lysine deacylase sirtuin 2

Sören Swyter, Matthias Schiedel, Daria Monaldi, Sándor Szunyogh, Attila Lehotzky, Tobias Rumpf, Judit Ovádi, Wolfgang Sippl, Manfred Jung

Research output: Contribution to journalArticle

9 Citations (Scopus)

Abstract

Sirtuins are NAD+ -dependent protein deacylases capable of cleaving off acetyl as well as other acyl groups from the ε-amino group of lysines in histones and other substrate proteins. They have been reported as promising drug targets, and thus modulators of their activity are needed as molecular tools to uncover their biological function and as potential therapeutics. Here, we present new assay formats that complement existing assays for sirtuin biochemistry and cellular target engagement. Firstly, we report the development of a homogeneous fluorescence-based activity assay using unlabelled acylated peptides. Upon deacylation, the free lysine residue reacts with fluorescamine to form a fluorophore. Secondly, using click chemistry with a TAMRA-azide on a propargylated sirtuin inhibitor, we prepared the first fluorescently labelled small-molecule inhibitor of Sirt2. This is used in a binding assay, which is based on fluorescence polarization. We used it successfully to map potential inhibitor-binding sites and also to show cellular Sirt2 engagement. By means of these new assays, we were able to identify and characterize novel Sirt2 inhibitors out of a focused library screen. The binding of the identified Sirt2 inhibitors was rationalized by molecular docking studies. These new chemical tools thus can enhance further sirtuin research.

Original languageEnglish
Article number20170083
JournalPhilosophical Transactions of the Royal Society B: Biological Sciences
Volume373
Issue number1748
DOIs
Publication statusPublished - Jun 5 2018

Keywords

  • Assays
  • Deacetylases
  • Epigenetics
  • NAD
  • Sirt2
  • Sirtuins

ASJC Scopus subject areas

  • Biochemistry, Genetics and Molecular Biology(all)
  • Agricultural and Biological Sciences(all)

Fingerprint Dive into the research topics of 'New chemical tools for probing activity and inhibition of the NAD<sup>+</sup> -dependent lysine deacylase sirtuin 2'. Together they form a unique fingerprint.

  • Cite this